For best optimization of therapy, it is helpful to predict the metastatic potential of any localized tumor at the time of diagnosis. This is particularly critcal for prostate cancer (PCa). The widespread application of PSA screening is leading to the detection of an increasing number of low grade and small PCas. Left untreated, some of these PCas may progress and become lethal. Many of them, however, may remain indolent for the rest of the patients' lives. The Gleason scoring (GS) system that is currently practiced to predict the possibility of PCa progression fails to predict the risk, when GS is d7. There is unmet medical need for a method for predicting likelihood of progression of these localized tumors without major changes in the current clinical practice. Hypothesis: There is accumulating evidence that production of reactive oxygen species within PCa cells lead to PCa progression and metastasis. We hypothesize that cancer cells with high cellular ROS will modify the structural protein filamentous actin (F-actin). Such modifications will make the cells flexible enough to squeeze through the gaps surrounding the tumor and invade the blood and lymphatic vessels for circulation. Some of these circulating cells with activated MMPs and modified F-actin will escape the endothelia to metastasize. Our goal is to develop an integrated prognostic test based on growing primary tumor cells obtained from routine prostate biopsies in micro-scale in vitro models of tumor microenvironments and then performing quantitative imaging of FAD fluorescence to determine specific metabolic activities that leads to PCa progression. We will validate this pathway biomarker(s) by correlating the data from patient PCa tissues with disease outcome. Our Specific Aims are: 1) To quantitate the difference between fluorescence properties of wild-type and genetically engineered PCa cell lines with varying levels of activation of the ROS generating pathway in a microfluidic mimetic of the basement membrane and endothelium capable of determining their relative invasive proper- ties, 2) To establish FAD fluorescence intensity and lifetime data collected from multiple prostate TMAs as prognostic markers by correlating the data with patient outcome in a retrospective clinical validation study. The observed changes of FAD fluorescence as an indicator of the enzyme activation will be confirmed by measuring the metabolite levels in the laser captured tissues from the corresponding tissue sections, 3) a) To prepare single cell suspensions of PCa cells from the tissue samples from informed, consented patients and analyze FAD fluorescence and their invasive properties in the microfluidic system as standardized in Aim #1, b) To create a TMA from PCa tissues obtained from the same patients and perform FAD fluorescence analysis as standardized in Aim #2, c) To laser capture cancer tissues from the same TMA sections and estimate the metabolites as standardized in Aim #2 and d) To clinically establish the markers by correlating the data collected in a-c with patient outcome in a prospective clinical study.